Mendelian susceptibility to mycobacterial disease (MSMD) is a syndrome predisposing individuals to severe clinical disease upon infection with weakly virulent mycobacteria. These patients are also susceptible to Salmonella and, in endemic areas, to tuberculosis. The genetic dissection of MSMD has revealed disease-causing germline mutations in nine genes, all of which encode proteins involved in IFN-?-mediated immunity. I recently discovered children with MSMD due to autosomal recessive ISG15 deficiency. ISG15 is known to be an interferon (IFN)-/-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice I identified granulocytes as a major source of secreted free ISG15, stimulating the production of IFN-? by lymphocytes, including NK cells in particular. Treatment of the patients' leukocytes with recombinant human ISG15 restored IFN-? production to normal levels upon challenge with BCG and IL-12. The main goal of the project proposed is to determine how ISG15 contributes to the induction of IFN-? in NK and T cells, possibly via a putative receptor (ISG15R), through the use of three different, complementary approaches. Aim 1 is to identify ISG15R and other downstream molecules controlling the NK cell response to ISG15, through the use of siRNA and proteomic screens. Training in these screens will build upon my expertise in immunology, genetics and bioinformatics. It will augment my research skill portfolio facilitating my transitionto an independent investigator. Aim 2 is to delineate the ISG15 signaling cascade, based on studies of the cellular responses of patients with mutations in key signaling pathways (STATs, MAPK, NF-?B, LUBAC, etc.) and the definition of ISG15-stimulated genes other than IFN-? by microarray and RNAseq analyses. Aim 3 involves a distinct approach where I mine our database of exomes from 100 MSMD patients, based on the hypothesis that some of these patients carry mutations in genes controlling lymphocyte responses to ISG15. Together, these studies will address the important question of the identity of the molecule to which ISG15 binds on NK/T cells, the signaling pathway essential for its function, the other genes induced or regulated by ISG15 and whether there are humans with mutations affecting the ISG15 responsive pathway.